Powering the ABC multidrug exporter LmrA: How nucleotides embrace the ion-motive force

Agboh, Kelvin; Lau, Calvin Ho-Fung; Khoo, Yvonne Siew Khoon; Singh, Himansha; Raturi, Sagar; Nair, Asha V.; Howard, Julie A.; Chiapello, Marco; Feret, Renata; Deery, Michael J.; Murakami, Satoshi; Veen, Hendrik W. van

doi:10.1126/sciadv.aas9365

Cited by 17 publications

(18 citation statements)

References 40 publications

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“…Our findings for MsbA and MsbA-MD are reminiscent of previous studies on LmrA-MD which demonstrated apparent ethidium-proton symport [57]. Most recently, we have extended these studies to full-length LmrA in cells and proteoliposomes, and used electrophysiological studies on LmrA in planar lipid bilayers and giant unilamellar liposomes to characterise the stoichiometry of the ion transport reaction in detail [58]. The efflux of one ethidium + and one H + by LmrA occurs in symport with one Cl -, in counter-transport to two Na + , in an electrogenic reaction that is driven by the inwardlydirected sodium motive force in cells (interior negative and low).…”

Section: Ion Transport Reactionssupporting

confidence: 78%

“…Although mutations in NBDs of LmrA and MsbA that inactivate ATP hydrolysis (e.g. deletion of lysine residue in Walker A in MsbA-ΔK382 and LmrA-ΔK388) inactivate ethidium transport in metabolically-active cells [53,56,58], both deletion mutants reconstituted in proteoliposomes are transport-active in the presence of appropriate electrochemical ion gradients. This suggests that, when nucleotide is present, nucleotide hydrolysis is compulsory for transport activity to prevent nucleotide trapping of LmrA.…”

Section: Ion Transport Reactionsmentioning

confidence: 99%

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Energy coupling in ABC exporters

Veen

Singh

Agboh

et al. 2019

Research in Microbiology

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Section: Ion Transport Reactionssupporting

confidence: 78%

Section: Ion Transport Reactionsmentioning

confidence: 99%

Energy coupling in ABC exporters

Veen

Singh

Agboh

et al. 2019

Research in Microbiology

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“…For example, Hoechst was used in studies of drug efflux-based antibiotic resistance in Salmonella enterica serovar Typhimurium 8,9 and Acinetobacter baumannii 10,11 , and in transport measurements for the ABC multidrug transporters LmrA from Lactococcus. lactis [12][13][14] , Sav1866 from Staphylococcus aureus 15 , MsbA and YbhFSR from E. coli 16,17 , and PatAB from Streptococcus pneumoniae 18 . Hoechst transport assays were used in studies of secondary-active multidrug and toxic compound extrusion (MATE) transporters VcmA from Vibrio cholerae 19 and AbeM from A. baumannii 20 , and in studies of a novel natural product inhibitor of the major facilitator superfamily (MFS) multidrug transporter NorA from Staphylococcus aureus 21 .…”

mentioning

confidence: 99%

Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP

Swain

Guo

Singh

et al. 2020

Sci Rep

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Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior. In transport assays, Hoechst 33342 (referred to as Hoechst) is a commonly used substrate, the fluorescence of which changes in the transport process. With three basic nitrogen atoms that can be protonated, Hoechst can exist as cationic and neutral species that have different fluorescence emissions and different abilities to diffuse across cell envelopes and interact with lipids and intracellular nucleic acids. Due to this complexity, the mechanism of Hoechst transport by multidrug transporters is poorly characterised. We investigated Hoechst transport by the bacterial major facilitator superfamily multidrug-proton antiporter LmrP in Lactococcus lactis and developed a novel assay for the direct quantitation of cell-associated Hoechst. We observe that changes in Hoechst fluorescence in cells do not always correlate with changes in the amount of Hoechst. Our data indicate that chemical proton gradient-dependent efflux by LmrP in cells converts populations of highly fluorescent, membrane-intercalated Hoechst in the alkaline interior into populations of less fluorescent, cell surface-bound Hoechst in the acidic exterior. Our methods and findings are directly relevant for the transport of many amphiphilic antibiotics, antineoplastic agents and cytotoxic compounds that are differentially protonated within the physiological pH range.

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“…13 μl for band 1 and 15 μl for bands 2-5 was loaded on QExactive for 1h runs. All LC-MS/MS experiments were performed using a Dionex Ultimate 3000 RSLC nanoUPLC (Thermo Fisher Scientific Inc, Waltham, MA, USA) system and a QExactive Orbitrap mass spectrometer (Thermo Fisher Scientific Inc, Waltham, MA, USA) as described recently [20]. The mass spectrometry proteomics…”

Section: In Gel Digestion and Mass Spectrometrymentioning

confidence: 99%

Proteomics of intracellular freezing survival

et al. 2020

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Panagrolaimus sp. DAW1, a nematode cultured from the Antarctic, has the extraordinary physiological ability to survive total intracellular freezing throughout all of its compartments. While a few other organisms, all nematodes, have subsequently also been found to survive freezing in this manner, P. sp. DAW1 has so far shown the highest survival rates. In addition, P. sp. DAW1 is also, depending on the rate or extent of freezing, able to undergo cryoprotective dehydration. In this study, the proteome of P. sp DAW1 is explored, highlighting a number of differentially expressed proteins and pathways that occur when the nematodes undergo intracellular freezing. Among the strongest signals after being frozen is an upregulation of proteases and the downregulation of cytoskeletal and antioxidant activity, the latter possibly accumulated before freezing much in the way the sugar trehalose has been shown to be stored during acclimation.

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Powering the ABC multidrug exporter LmrA: How nucleotides embrace the ion-motive force

Abstract: Researchers study how different forms of metabolic energy are coupled to drug extrusion by an ATP-binding cassette transporter.

Cited by 17 publications

References 40 publications

Energy coupling in ABC exporters

Energy coupling in ABC exporters

Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP

Proteomics of intracellular freezing survival

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